This project seeks to understand the molecular dynamics of the allosteric transition in hemoglobin, using spectroscopic probes of protein structure to investigate the change from low- to high-affinity states. These states control the hemoglobin function in healthy and diseased states. Their understanding is also critical for the development of hemoglobin-based blood substitutes. The spectroscopic techniques to be applied to hemoglobin are ultraviolet resonance Raman spectroscopy, and Fourier transform infrared spectroscopy. Both will be used in a time-resolved mode to generate spectra of transient states of the protein, and thereby provide structural mapping of the allosteric reaction coordinate. Isotope labeling of recombinant protein, and site-directed mutagenesis will be used to increase the power of these techniques. The lessons learned from hemoglobin will help to understand how proteins manage allosteric transitions.